The theme of our investigations has been that the molecular pathways of programmed cell death (PCD) may be relevant to the pathogenesis of Parkinson's disease and allied disorders. In recent years it has also become apparent that PCD regulates viability in cell-based therapeutic approaches, including tissue implants and neural stem cells. There has been tremendous growth in our knowledge of the molecular basis of PCD. However, most of this knowledge derives from relatively simple in vitro systems. While there are universal aspects of PCD mechanisms, it is nevertheless also clear that PCD is context dependent. It is therefore essential to translate this new knowledge to the in vivo context. A unique aspect of our approach is to examine PCD in postmitotic, phenotypically defined dopamine (DA) neurons in living brain. We will investigate two themes related to regulation and effector mechanisms of cell death in these neurons. While there is evidence that natural cell death (NCD) in DA neurons is regulated by striatum-derived neurotrophic support, the nature of these factors remains unknown. Theme 1 will examine the possibility that GDNF or neurturin (NTN) may serve as such factors. Our first Aim will examine the effect of increased striatal expression of GDNF, in a unique temporally-regulated bi-transgenic model, on the mature number of DA neurons. The second Aim will determine whether endogenous striatal GDNF regulates the magnitude of NCD in DA neurons, through the application of "knock down" approaches. Our third Aim will directly compare the potency of GDNF and NTN to suppress apoptotic death in a developmental axotomy model. Theme 2 will seek to identify important proteases mediating PCD in DA neurons in vivo. We have shown that activated caspase-3 is expressed in apoptotic DA neurons. In Aim IV, we will examine the functional significance of its expression, by studying the magnitude and protein cleavage characteristics of NCD, and the size of DA progenitor pools, in caspase-3 null animals. Many in vitro studies have shown that caspases-independent pathways exist. In Aim V, we will examine expression of the proteasome complex in PCD in DA neurons in vivo. The new knowledge gained by the studies outlined in this application will have direct implications for concepts of pathogenesis of Parkinson's disease and for approaches to optimizing cell-based treatments.